The overall goal of this proposal is to continue to explore a novel photochemical method for killing antibiotic resistant pathogenic bacteria or fungi in models of localized infection. Photodynamic therapy (PDT) employs a non-toxic dye termed a photosensitizer (PS) and low intensity visible light, which in the presence of oxygen produce cytotoxic species that damage proteins, lipids and nucleic acids and kill cells. PDT has the advantage of dual selectivity in that the PS can be targeted to its destination cell type or tissue, and in addition the illumination can be spatially directed to the area of infection. In the previous funding period we established that polycationic delivery vehicles such as poly-L-lysine could be conjugated to PS such as chlorin(e6), and these molecular delivery vehicles for PS increased the selective binding to bacteria and enabled the PS to penetrate the cell walls of Gram (-) bacteria to dramatically potentiate light-mediated killing.. We used luminescent bacteria and a low-light imaging camera to demonstrate that PDT will kill both Gram (-) species (eg Pseudomonas aeruginosa) and Gram (+) species (eg Staphylococcus aureus) in vivo in animal models of wounds, burns and deep established infections. Localized PDT may have an additional advantage in that it is also possible to inactivate secreted extracellular virulence factors that pathogenic bacteria use to establish infections and invade tissue. This competing renewal will seek to explore new ways of increasing the potency and applicability of antimicrobial PDT. Four specific aims will focus on (1) studying the photochemical mechanisms of photodynamic inactivation of microbes (that may be very different from cancer cells) with the aim of devising simple combination treatments;(2) investigating the new discovery that low non-toxic concentrations of hydrogen peroxide dramatically potentiate antimicrobial PDT by orders of magnitude;(3) synthesizing and testing a third generation polycationic PS conjugates with quaternized amino groups that retain cationic character under all conditions;(4) testing the above treatments in mouse models of acute or chronic wounds and burns infected with pathogenic bacteria (P. aeruginosa or S. aureus), together with an entirely new model of spectrally resolved fluorescence imaging of GFP Candida albicans or Aspergillus fumigatus growing in traumatic lesions in various strains of mice. These avenues of research are expected to suggest simple procedures to optimize antimicrobial PDT and hasten its wide introduction into clinical practice. PUBLIC HEALTH RELEVANCE The alarming rise in prevalence of antibiotic resistance amongst pathogenic bacteria has led to worries that previously treatable infections could soon be incurable. Traumatic or surgical wounds and burns are common sites of infection that can progress to sepsis and death if they fail to be controlled by antibiotics. Photodynamic therapy (PDT) involves a combination of non- toxic dyes and harmless visible light that in combination produce highly toxic reactive oxygen species. If the dye is targeted to the bacterial cell PDT can be a highly effective local antimicrobial therapy with little damage to host tissue. This application seeks to determine the optimum parameters for antimicrobial PDT and will look at new synergistic combination therapies.